Mushrooms have been valued by humankind as a source of nutrition, medication and numerous other purposes in the far eastern and recently in the western world. Mushrooms are an immensely rich source of biologically active secondary metabolites. Secondary metabolites are generated as a response to external stimuli such as nutritional or climatic changes. Usually, they are accumulated in only certain parts of the mushroom. The secondary metabolites produced by mushrooms exhibit largely diverse structural differences. Therefore, the isolation and separation process can be lengthy and tedious. However, isolation of natural products usually combines various separation techniques, which depend on the solubility, volatility and stability of the desired compound to be separated. Hence, the choice of different separation steps is of greater significance and an analytical-scale optimization of the separation parameters is worthwhile to make the isolation process shorter and convenient.
The first step in the process of obtaining secondary metabolites from biogenic materials is to extract them from the cellular matrix by means of extraction. The choice of extraction method is of greater importance due to the complex composition of the material and the minute amounts of the constituents available. An incorrect choice will cause the entire isolation process failed. In such cases, some or all of the targeted components of the specimen cannot be released satisfactorily from the matrix. Thereby, it is essential to select a suitable extraction method to obtain the total extract which is referred to as the crude extract. A logical next step in the isolation is to separate the desired components from the crude extract. This can be accomplished by liquid-liquid partition or by some low-resolution chromatographic isolation including normal phase column chromatography and size-exclusion chromatography. The goal of these steps is to concentrate the components of interest in order to facilitate the final purification steps. The third phase in the isolation usually involves high-resolution method to separate the desired compounds from the other components which remain in the extract. Since undesired components of the extract are likely to bear some closeness to the targeted compounds, requisite for optimization of the separation method become an important concern to accomplish sufficient resolution in the final preparative isolation. Frequently, the final isolation step involves liquid chromatography, preferably, high-pressure liquid chromatography (HPLC), counter-current chromatography (CCC), droplet counter-current chromatography (DCCC), rotation locular counter-current chromatography (RLCC), centrifugal partition chromatography (CPC).
In current invention, a novel and easy isolation method has been optimized to isolate inoscavin A compound of formula 1 from the Sri Lankan mushroom Fulviformes fastuosus to get rid and overcome above difficulties encountered during a separation and isolation process. Being a tropical country, Sri Lankan biota has enormous fungal diversity and consists of a variety of macrofungi species with medicinal and aromatic values. Although, some of the species are used in traditional medicine, most of them have been still not discovered for their medicinal values. Therefore, it is worthwhile to investigate the bioactive properties of unexplored mushrooms in order to discover medicinally important compounds of natural origin. Hence, this study is focused on antiproliferative activity of an unexplored macrofungus, Fulviformes fastuosus which is harvested from the dry zone forest reserves in Sri Lanka. Yet there are no reports available on bioactive properties of this species. F. fastuosus is a terrestrial basidiomycete which belongs to the family hymenochaetaceae. Majority of the species in this family are of medicinal value, while some are plant pathogens causing a white rot.
The last decade has witnessed the overwhelming interest of western research fraternity in pharmaceutical potential of mushrooms. Constituent molecules of mushrooms organelles and secondary metabolites have long been believed to possess pharmacologically important properties including antioxidant, anticancer, immunomodulatory, anti-inflammatory and anti-diabetic properties. There are approximately 650 species of macro fungi that have been reported to possess anticancer activity. The anticancer activity of the macrofungi was first reported for the extracts of fruiting bodies of Boletus edulis against the sarcoma 180 line in mice. The mounting evidences from various scientific studies across the world, regarding antitumor application of mushroom extracts and substances unarguably make it a fast-track research area worth mass attention. In spite of the availability of novel antineoplastic agents, cancer remains as the second leading cause of death affecting millions of people per year. The recent cancer therapies such as radiotherapy, chemotherapy and hormonal therapy have been made a modest progress in reducing the morbidity and mortality caused by cancer to the expected level and also produce unpleasant side effects. Hence, there is a recent upsurge in the interest of natural sources including mushrooms due to their promising anticancer properties. Molecules derived from medicinal mushrooms play a dominant role in the discovery and development of effective drug leads for cancer. The antitumor compound calvacin was the most commonly used natural product isolated from a mushroom. It was isolated from the giant puffball Calvatia gigantean and found activity against many experimental tumors, including mammary adenocarcinoma 755, sarcoma 180, leukemia L-1210, and HeLa cell lines. Moreover, Lentinan, schizophyllan and krestin are the natural products isolated from Lentinus edodes, Schizophyllum commune, Trametes versicolor mushrooms respectively. Currently, they have been approved in Japan as prescription drugs for the treatment of cancer. Ganoderma lucidum is also an important medicinal mushroom used today, acclaimed as “mushroom of immortality”. Polysaccharide (GLPS) fractions isolated from G. lucidum have been reported to possess strong antitumor effect.
Inoscavin A flavonoid isolated from Phellinus species via a different isolation method have demonstrated in vitro inhibition of cell proliferation against HepG2, MGC80-3, HCT-116, HeLa, MCF-7 and A549 cancer cell lines. In current invention, inoscavin A has been isolated from an unexplored mushroom, Fulviformes fastuosus using a novel and convenient method and composition comprising inoscavin A thereof to treat rhabdomyosarcoma cancer conditions. In vitro cytotoxicity of the inoscavin A has been tested against rhabdomyosarcoma (RMS) using RD cell line. RMS is the most common soft tissue malignancy in childhood and soft tissue sarcomas (STS) constitute about 7% of all malignancies in children and adolescents under the age of 20 years. RMS is characterized by a high grade of malignancy, local invasiveness and a marked tendency to metastasize, while generating good response to chemotherapy and radiotherapy. RMS accounts for about 40% of pediatric soft tissue malignancies. Approximately 65% of cases diagnosed in United States have been identified as RMS in children less than six years old and approximately 250 new cases are detected each year in the USA. The incidence of RMS in Iraq is about 3% of childhood cancer cases less than 14 years of age in 2010. RMS is arisen from skeletal muscle precursors and divides in to two major subtypes, embryonal and alveolar, which differ prominently in their outcomes. Embryonal RMS usually occurs in children less than 10 years old with 5 year survival rate which is closer to 75%. Alveolar RMS presents throughout childhood and adolescence and is accompanying with poor prognosis with a 5 year survival rate below 50%. Prognosis of the patients with obstinate or relapsed RMS is worse, displaying lower survival rates ranging from 10 to 30%.
Different RMS cell lines can be used in basic research and preclinical testing for RMS. RD cells are one of the most commonly used cell line in RMS research. Therefore, RD cell line is used in the current study to determine the anticancer effect of inoscavin A. RD cell line is derived from a biopsy of 7 year old child having pelvic refractory RMS, previously treated with cyclophosphamide and radiation. It has been found that RD exhibited growth inhibition to cyclophosphamide and vincristine. Tolfenamic acid was also tested against RD cell line and showed decreased tumor size and reduction of cell migration after treatment. However, limited number of agents including natural sources has been tested yet against RMS. Current invention is ultimately targeted the investigation of anticancer activity of isolated compound inoscavin A against RMS and found that it is capable to induce apoptosis of RD cells. In addition, antiproliferative activity of inoscavin A against normal mammalian cell line (CC-1) was also investigated to compare the toxicity produced by inoscavin A against cancerous and normal cells by determining the selectivity index (SI). Selectivity index or therapeutic index is defined as the relative effectiveness of the investigational product in inhibiting tumor cell growth compared to inducing cell growth of normal cells. It is evaluated by the ratio of CC50/IC50. Inoscavin A, tested against RD cells exhibited an extremely large value for therapeutic index giving maximum antiproliferative activity for cancer cells with minimal toxicity for normal cells. Thereby, inoscavin A isolated from above described method can be effectively developed as a medicament for RMS. To the best of our knowledge, this is the preliminary and novel record that a natural product isolated from a mushroom being tested against rhabdomyosarcoma possesses potent anticancer effect.